Seroprevalence and risk factors for COVID-19 in the metropolis of the Brazilian Amazon

The devastating effect of health system overload was observed after cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) peaked in April 2020 in Belém, a capital metropolis in the Brazilian Amazon. Our results showed a high seroprevalence (39.24%) of anti-SARS-CoV-2 IgG antibodies among the population of the capital of the state of Pará after the first wave of the pandemic. Old age, mixed race, a high school education level, low income and contact with infected individuals are risk factors and may be driving seropositivity and exposure to SARS-CoV-2 in this population. This study is one of the first to provide important information to understand the socioeconomic inequalities, behavioral characteristics and viral transmission dynamics associated with the risk of SARS-CoV-2 infection in the capital of the state of Pará, northern Brazil.

Individuals already vaccinated with one or two doses against SARS-CoV-2, those who did not complete the epidemiological questionnaire, those who did not sign the informed consent form and those younger than 7 years were excluded from the study.
Data collection. After obtaining a signed informed consent form for participation in the study, individuals were interviewed using a structured questionnaire addressing clinical, demographic and behavioral characteristics as possible risk factors for SARS-CoV-2. We obtained signed informed consent forms from individuals aged 18 years or older. Children aged 7-11 years and adolescents aged 12-17 years signed a free and informed assent form, and their parents or guardians signed an informed consent form.
The following individual data were collected: (1) sociodemographic data-age, sex, income, ethnicity and marital status; (2) comorbidities; (3) behavioral information on preventive measures, such as mask use, travel, hand hygiene, social distancing and contact with people infected with SARS-COV-2; and (4) information related to COVID-19-the presence of symptoms, previous diagnosis, self-medication and medication prescribed to treat symptoms, hospitalization and the need for mechanical ventilation. The participants' data were recorded using EPI Info™ software version 7.2.4. 10 and stored in the local server of the Virology Laboratory of UFPA.
Sample collection, processing and storage. After questionnaire completion and collection of signed informed consent forms, venous blood samples (10 ml) were collected from the volunteers by venipuncture into a vacuum tube containing EDTA. Subsequently, the samples were processed anonymously in the Virology Laboratory; plasma and leukocytes were isolated and then stored at − 70 °C.

Serological analysis.
Anti-SARS-COV-2 IgG antibodies were detected by ELISA (Euroimmun, Lübeck, Germany), which uses the recombinant structural protein (S1 domain) of the S protein as an antigen, following the manufacturer's protocol. The samples were classified as nonreactive (ratio < 0.8), indeterminate (0.8 ≤ ratio ≤ 1.1) or reactive (ratio > 1.1) for IgG, as suggested by the manufacturer. The manufacturer reported a clinical sensitivity of 75-93.8% (> 10-20 days to ≥ 21 days after disease onset) and a specificity of 99.6% for IgG antibodies.
Statistical analysis. The seroprevalence estimated based on the sociodemographic characteristics of the population is presented as a count and a percentage with a 95% confidence interval (CI), which were calculated in the program BioEstat 5.0. Correlations between symptoms and positivity were estimated using BioEstat 5.0. Associations between the presence of anti-SARS-CoV-2 IgG antibodies and the study variables were estimated using univariate analysis with the chi-square test or G test. A significance level of 5% (p < 0.05) was established. Uni-and multivariate logistic regression analyses were used to explore associations between risk factors (sociodemographic and behavioral characteristics and symptoms) and SARS-CoV-2 infection and between symptomatic and asymptomatic cases and the presence of antibodies. The definitions established by the Brazilian Ministry of Health were used as a classification criterion for symptomatic individuals 11 . Cases that did not meet these criteria or individuals who did not show any symptoms were classified as asymptomatic. All univariate and multivariate analyses were performed using Minitab software, version 14.0. Graphs were generated in GraphPad version 7.0 and Excel 2010.
Ethics approval and consent to participate. The study was submitted to and approved by the National Research Ethics Committee (CONEP, acronym in Portuguese) and the Human Research Ethics Committee of the Health Sciences Institute of Federal University of Pará (UFPA) under CAAE n. 31800720.1.0000.0018 in compliance with the guidelines and regulatory standards for research involving human beings, with all methods conducted in accordance with the Declaration of Helsinki.

Results
Socioeconomic and behavioral characteristics and comorbidities associated with the risk of SARS-CoV-2 infection.

Discussion
We estimated the prevalence of anti-SARS-CoV-2 IgG antibodies for the S1 subunit of the spike glycoprotein and factors potentially associated with SARS-CoV-2 infection 6 months after the beginning of the first wave of COVID-19 in Belém, Pará, Brazil. To our knowledge, this is one of the first seroepidemiological studies describing the prevalence of anti-SARS-CoV-2 antibodies in the city of Belém. The seroprevalence of anti-SARS-CoV-2 antibodies varies based on geographic region, study population, sampling time, laboratory method and COVID-19 incidence at the sampling site. In Brazil, the prevalence of antibodies was highly heterogeneous when considering the five regions of the country, with the North and Northeast regions clearly having the highest seroprevalence rates, which were strongly associated with low socioeconomic   www.nature.com/scientificreports/ indicate a seropositivity of 39.24% in the city of Belém. We believe that the seroprevalence in Belém may be even higher than that observed when we consider our previous results of a follow-up of a post-COVID-19 cohort in which we found a 30% loss of anti-SARS-CoV-2 IgG antibodies 3 months after COVID-19 diagnosis 17 . The choice of serological test was a determining factor for the prevalence rate of antibodies found in this study because due to good sensitivity and specificity, the test allowed the detection of asymptomatic seropositive individuals who usually do not seek medical care or undergo diagnostic tests and are therefore underreported in official numbers. According to the Brazilian Institute of Geography and Statistics, the population of the capital city of Belém is 1.5 million inhabitants 18 . Based on our seroprevalence results, which indicated that 39% of participants had been exposed to SARS-CoV-2, the estimated number of infected individuals in the capital would be 585,000, which is 10 times higher than the number reported by the Municipal Department of Health as of December 30, 2020 19 , as these reports are based only on cases of symptomatic individuals seeking diagnosis and treatment services. We believe that the seroprevalence results observed in the present study are not influenced by possible false-positive or false-negative results, since a methodology with high sensitivity and specificity was used.
Our study failed to identify a significant difference between the sexes regarding the risk of SARS-CoV-2 infection, which is similar to findings reported in other seroprevalence studies 20,21 . The presence of comorbidities was not a predictor of positivity for anti-SARS-CoV-2 IgG antibodies, which is similar to observations in another study 22 . However, comorbidities may be associated with a high risk of serious complications and death due to COVID-19 23,24 . The age group most exposed to the virus and with the highest seroprevalence rates was adults aged over 70 years, which is similar to observations in other studies 25,26 . As home lockdown was voluntary, the recommendations for social lockdown may not have been adequately followed, suggesting exposure at the family level. The antibody rate by age can be a useful tool to track vaccine prioritization for the population, as defined by the National Plan for the Operationalization of Vaccination against COVID-19 of the Brazilian Ministry of Health 27 .
Individuals who self-reported being mixed race have a high prevalence of antibodies and a high risk of viral infection because they represent the majority of the Brazilian population. Our results demonstrate a robust association between socioeconomic conditions and the risk of SARS-CoV-2 infection, with a higher risk for individuals with a high school education and with a family income less than or equal to one or two minimum wages-socioeconomic characteristics that are prevalent in low-and middle-income countries such as Brazil. Family income was the most significant risk factor. Families with incomes between three and five times the minimum wage were less likely to be infected than were families with lower incomes, which is similar to the rest of the country 12,13,28 . Socioeconomic inequalities became even more evident with the pandemic. We assume that social health differences contributed to these initial observations and resulted in differential exposure to the virus and differential vulnerability to infection, indicating the need for preparedness plans for vulnerable communities to ensure a rapid and coordinated response to protect these groups from future pandemics or crises at an early stage.
Regarding infection prevention measures, interestingly, we found that individuals who regularly used a mask and frequently engaged in hand hygiene had the highest seroprevalence rates. Notably, our data suggest that the lack of information on proper handwashing techniques, proper mask use and mask efficiency explain the high prevalence of IgG anti-SARS-CoV-2 antibodies in this population. Our data indicate that direct contact with individuals infected with SARS-CoV-2 increases the risk of infection, which is consistent with results reported in other studies 29,30 .
Our results indicate a relatively high seroprevalence of asymptomatic SARS-CoV-2 infection (37.5%) in the city of Belém, corroborating other studies conducted in Brazil 16,31 , which complicates disruption of the transmission cycle, since a high percentage of individuals who claimed to have had no symptoms of COVID-19 were seropositive, thus highlighting the possibility of silent transmission of the virus by asymptomatic individuals. www.nature.com/scientificreports/ Mass testing is important for rapid identification of asymptomatic cases and isolation of infected individuals, with testing and monitoring of their contacts, to prevent the spread of the virus 32 . Clinical symptoms such as fever, body aches, loss of smell, loss of taste, shortness of breath and fatigue are strongly associated with SARS-CoV-2 infection, with studies showing symptom profiles similar to those found in our population. In March 2020, the first reports relating the loss of smell and/or taste with COVID-19 were published 33,34 . The available data suggest a prevalence of loss of smell and/or taste ranging from 31 to 85% among patients with COVID-19 [33][34][35][36][37] . Our results support that otorhinolaryngological symptoms are prevalent among patients with mild to moderate COVID-19 infection because we observed a high prevalence of these symptoms in participants with mild or moderate infection and positive results for anti-SARS-CoV-2 IgG antibodies.
In Belém, we observed that individuals aged 40-59 years had a higher risk of developing symptoms than younger people. A retrospective cohort study found that most hospitalized patients with COVID-19 symptoms had a mean age of 54 years and that advanced age was a risk factor for death among adults 38 . The effects of age on immune system function, the activity of T and B lymphocytes and excess production of pro-inflammatory cytokines influence the onset of symptoms owing to poor control of viral replication 39 . Thus, the correlation between age and the risk of SARS-CoV-2 infection or the risk of developing critical illness observed in this study may be essential information for planning health resources to ensure the sustainability of the system.
All countries are urged to make increasingly rapid decisions regarding socioeconomic issues and public health policies to contain the pandemic. High-quality data, as presented here, are necessary to facilitate decision making. This study provides important information about the high prevalence of SARS-CoV-2 in the city of Belém and indicates that individuals with low socioeconomic status and/or little education are more susceptible to infection-a pattern commonly found in low-income countries. We also discovered the impact of the proper use of prevention and control measures and suggest that preventive measures are essential to minimize symptomatic and asymptomatic transmission in the community. Seroprevalence surveys will be needed to identify dynamic trends and monitor disparities to profile future transmission dynamics and formulate protective responses for vulnerable groups.
We understand that the present study has some limitations. We encountered difficulties when obtaining samples due to the lockdown and social distancing decrees related to the pandemic, which may have led to a sample bias in the statistical analysis. Nevertheless, our analyses have strengths. The study population was representative of both sexes, all ages and different economic groups and races. The serological test based on the S protein can detect past infections, including asymptomatic and recovered cases, providing better information on the prevalence of the disease in the population. To date, this is one of the first epidemiological studies to investigate risk factors for SARS-CoV-2 infection in the city of Belém, northern Brazil.
All countries are urged to make increasingly rapid decisions regarding socioeconomic issues and public health policies to contain the epidemic. We were able to estimate the high seroprevalence of SARS-CoV-2 in the first six months after the first wave of COVID-19 in the city of Belém and identify the factors that may be driving seropositivity and exposure to SARS-CoV-2 in this population. These findings highlight the importance of serosurveillance for estimating the real impact of the COVID-19 pandemic, identifying trends in viral transmission and monitoring population disparities to profile future transmission dynamics and formulate protective responses for vulnerable groups. In addition, these results may be essential for monitoring the evolution of the pandemic after easing social distancing measures and the implementation of a vaccination schedule in Brazil as a basis for other countries.

Data availability
All data supporting the findings of this study are available in the article.